Field of the Invention
The invention relates to a method for detecting a nucleotide sequence. It furthermore relates to a kit for carrying out the method.
Such a method is known by the term xe2x80x9cpolymerase chain reactionxe2x80x9d (PCR). PCR is suitable for the generation of large amounts of a desired DNA sequence. To carry out the method, two synthetic oligonucleotide primers which are complementary to sections on a strand and on a counter-strand which flank the desired DNA sequence, are added to a solution comprising the sample. After amplification, the desired DNA sequence can be detected by addition of detection reagents, for example by means of a color reaction or electrophoresis.
The known PCR method requires a long reaction time, owing to necessary diffusions in the solution. Moreover, the detection of the PCR product formed, for example by means of color reaction or electrophoresis, is time consuming. Moreover, it is necessary, for detection, to open the reaction batch comprising the multiplied products. This constitutes a risk of contamination. Finally, the sensitivity of the known method is insufficient for certain applications.
WO 94/02636 describes a method in which a first primer bound to a solid phase is brought into contact and hybridized with a sample comprising a nucleotide sequence. Thereupon, the sample container is opened, and the sample is treated with a second primer, whereupon the nucleotide sequence hybridizes. The second primer is labeled. It accumulates on the solid phase, where the label can be observed. Furthermore, it is known from WO 94/02636 to carry out a polymerase chain reaction using three primers in solution, the solution being brought into contact successively with the primers.
The disadvantage of the methods disclosed in WO 94/02636 is that the reaction vessel must be opened in order to bring the sample into contact with the second and, if appropriate, the third primer. This may result in contaminations.
WO 96/26291 describes a method for distinguishing between several alternative DNA sequences. In this method, a first and a second primer are bound to a solid phase. A third primer is in solution. This method is only suitable for distinguishing between the presence of alternative DNA sequences, but not for detecting a particular nucleotide sequence. In the PCR, only two primers are included in the reaction, one of which is bound to a solid phase. This primer shows a low degree of mobility and accessibility. The method is less suited for PCR than when free primers are present.
A method for the specific detection of DNA sequences is known from Chemical Abstracts 126: 70853z (1997) re: chapture [sic] PCR amplification with single-sided specifity across mutation break-points; Lagerstroem-Fermer, Maria et al; Lab. Protoc. Mutat. Detect. 1996, 183-188. In this method, the DNA sequences to be detected are first amplified and then bound to a solid phase. In subsequent steps, the DNA sequence to be detected is detected by further PCRs. In this method, the DNA sequence to be detected is immobilized on the solid phase by binding.
The use of synthetic residues in primers which cannot be replicated is known from Chemical Abstracts 120: 155091a (1994): Incorporation on nonbase residues into synthetic oligonucleotides and their use in the PCR; Gade et al.; Genet. Anal.: Tech. Appl. 1993, 10(2), 61-5. In the PCR, double-stranded DNA is produced by the polymerase until the latter encounters such a synthetic residue. From this position on, the DNA strand remains single-stranded. The single-stranded DNA sequence is capable, without previous denaturation, of hybridizing with a complementary, further DNA bound to a solid phase.
The fact that a fluorescence energy transfer is possible between fluorogenic groups bound to oligonucleotides when these fluorogenic groups are fixed at defined intervals by a hybridization is known from Cardullo R. A. et al: Detection of nucleic acid hybridisation by nonradiative fluorescence resonance 15 energy transfer; Proc. Natl. Acad. Sci. USA 85 (1988) 8790-8794.
A method for detecting specific DNA in which the sample is initially subjected to a first amplification by means of PCR is disclosed in WO 90/11369. Thereupon, the sample is transferred into another reaction vessel, where it is brought into contact with a second primer, which is bound to a solid phase. A second amplification by means of PCR takes place. Again, this method suffers the disadvantage of a risk of contamination when transferring the sample from the first into the second reaction vessel.
The object of the invention is to eliminate the disadvantages of the prior art. In particular, it is intended to state a rapid method for detecting a nucleotide sequence with a reduced risk of contamination.
This object is achieved within the scope of the invention by a method for detecting a nucleotide sequence by means of polymerase chain reaction, where the nucleotide sequence is a constituent of a double-strained nucleic acid molecule which is in solution and which is comprised of a strand (S) and a counter-strand (G), the method comprising the following steps:
a) adding a first primer to the solution, where the first primer is complementary to a first segment of the strand located at the 5xe2x80x2-terminus of a central segment, and a second primer, where the second primer is complementary to a 5xe2x80x2-terminal second segment of the counter-strand,
b) bringing the solution into contact with a third primer whose 5xe2x80x2-terminal end is bound to a solid phase, where the third primer is complementary to the central segment of the strand comprising the nucleotide sequence,
c) closing a reaction vessel which contains the solution,
d) repeatedly heating and cooling the solution with the following steps:
d1) heating to 92xc2x0 C.-100xc2x0 C.
d2) cooling to 40xc2x0 C.-600xc2x0 C.
d3) heating to 72xc2x0 C.-75xc2x0 C. and
e) detecting the nucleic acid molecule during or after step lit. [sic] d) with a closed reaction vessel.
This concentrates the desired nucleotide sequence on the solid phase. The reaction time required is shortened and the sensitivity improved.
The third primer is advantageously a DNA molecule or a PNA/DNA chimera.
The solid phase is a polymer, preferably an electrically conductive polymer, which may comprise a polycarbonate, trimethylthiophene, triaminobenzene and/or a polycarbene and/or carbon fibers. The solid phase is expediently a microtiter plate in whose well the third primer may be bound covalently or by means of biotin.
In an especially advantageous embodiment feature, an interaction allowing a nonradiative or direct energy transfer between a first and a second fluorophoric molecule is generated or removed when the nucleotide sequence is present. The first fluorophoric molecule may be bound to the solid phase, or else the first fluorophoric molecule is bound to the solid phase via the third primer.
A particularly simple variant consists in the third primer having a hairpin loop and the first fluorophoric molecule being bound to a first loop segment and the second fluorophoric molecule opposite to a second loop segment at a distance which allows the interaction to take place. The interaction can be removed by hybridization with a counter-strand which is complementary to the third primer or by a synthesis taking place on the third primer. In this context, the second fluorophoric molecule may be bound to the second primer. The abovementioned features allow simple and automatable detection of the nucleic acid molecule as early as during the PCR.
The invention furthermore provides a kit for carrying out the method according to the invention, comprising, as first component, a first primer and as second component a second primer, and also a third primer whose 5xe2x80x2-terminal end is bound to a solid phase.
The solid phase is expediently a microtiter plate. As further components, the kit may comprise the buffers required for carrying out the polymerase chain reaction, the deoxynucleotide triphosphates required for multiplying the nucleotide sequence, and the polymerase required for carrying out the polymerase chain reaction, preferably a Taq polymerase.
The first primer and the second primer and also the further components may be present in the lyophilized state. Thus, a reaction may be started by adding the solution comprising the nucleotide sequence. Alternatively the first and the second and/or the further components may be coated with wax. This allows the reaction to be started by heating the solution comprising the nucleotide sequence and the coated components.
Advantageous embodiments of the invention are illustrated in greater detail hereinbelow with reference to the in the drawing [sic]. The figures show: